The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.
One of the major causes of hepatitis are specific hepatitis viruses. Among them, HCV is now known to cause most cases of what was previously termed non-A, non-B (NANB) hepatitis, and causes the vast majority of post-transfusion and sporadic NANB hepatitis. HCV infections is generally symptomatically mild. However, at least half of all infected individuals appear to develop chronic hepatitis, and 20% of these may develop cirrhosis.
HCV is a small RNA virus containing a single molecule of RNA of about 10,000 nucleotides in length. The genome of HCV contains a single, long, open reading frame (ORF) that is translated into a single, large polyprotein and subsequently processed, and exhibits a large degree of nucleic acid sequence heterogeneity. The entire genome of HCV has been cloned and sequenced. To date, at least five related genotypes of the virus have been found. The relative abundance of each genotype throughout the world is the subject of intensive study. The 5′ untranslated region (UTR) of the virus is remarkably well conserved and has provided an excellent site for oligonucleotide probes and primers (Bukh et al., Proc. Natl. Acad. Sci USA 88:942-946, 1992).
Since multiple HCV subtypes exist with varying amino acid sequences, these subtypes vary geographically and play a role in disease virulence. HCV can also alter its amino acid pattern over time in an infected person, hampering vaccine development. Since most cases of hepatitis C are subclinical, even in the acute stage, hepatitis C is often uncovered by the serendipitous detection of anti-HCV in apparently healthy persons.
HCV causes at least 80% of post-transfusion hepatitis cases and a substantial proportion of sporadic acute hepatitis cases. It is also implicated in many cases of chronic hepatitis, cryptogenic cirrhosis, and hepatocellular carcinoma unrelated to HBV. Infection is most commonly acquired via blood, either from transfusion or IV drug use. Sexual transmission and vertical transmission from mother to infant can occur but are relatively rare. A small proportion of seemly healthy persons are chronic HCV carriers, who often have subclinical chronic hepatitis or even cirrhosis. HCV is associated with many disorders including “immune” disorders, such as glomerulonephritis.
Diagnosis of hepatitis C is based on the presence of serum antibody (anti-HCV), which is not protective and implies active infection. First generation serologic tests were often falsely positive, but second and third generation tests are more reliable. Anti-HCV often appears several weeks after acute infection, so a negative test does not exclude recent infection.
The measurement of HCV nucleic acid in serum has become an important tool to identify individuals with high viral replication, to monitor patients on therapy, and to predict whether antiviral therapy will be successful.
Several tests have been employed to detect HCV in serum and other body fluids. Hybridization assays for detecting HCV polynucleotides are known in the art. For example, Matthews and Kricka, Analytical Biochemistry 169:1, 1988; Landegren et al., Science 242:229, 1988; Mattlin, Clinical Chem. 35: 1819, 1989; and U.S. Pat. No. 4,868,105.
To increase the sensitivity of such assays, HCV nucleic acid sequences can be detected by reverse transcribing HCV genomic RNA to form cDNA, amplifying the resulting cDNA by, for example, the polymerase chain reaction (PCR), and detecting the presence of amplified product. The HCV detection assays based on PCR amplification of HCV polynucleotide sequences were described in U.S. Pat. No. 5,527,669; European Patent Publication No. 529,493; Young et al., J. Clin. Microbiol. 31(4): 882-886, 1993; and Young et al., J. Sin. Microbiol. 33(3): 654-657, 1995. Therefore, when active disease is suspended or post-treatment follow up is desired, detection of HCV-RNA by PCR provides a sensitive technique for the direct detection of HCV-RNA in patient serum.
Because of the sequence heterogeneity among HCV strains, there is still a need for primer oligonucleotides for amplifying HCV sequences, each chosen from a conserved region so that all, or almost all, strains will be amplified. U.S. Pat. No. 5,837,442, which is incorporated herein by reference, provides primers for the amplification of HCV nucleic acid with significantly high efficiency. This patent provides that amplification of HCV RNA can be carried out using a combined reverse transcription-polymerase chain reaction (RT-PCR) amplification, in which a single enzyme catalyzes the primer extension both from the initial genomic RNA template (i.e. reverse transcription) and from the DNA templates synthesized in the amplification process.
Hybridization assays for detection of nucleic acids are described in, for example, U.S. Pat. Nos. 6,258,569; 6,030,787; 5,952,202; 5,876,930; 5,866,336; 5,736,333; 5,723,591; 5,691,146; and 5,538,848. Publications for detection of HCV using Real-time PCR (Taqman systems) include the following: Henning et al., Transfusion 41(9): 1100-6, 2001; Meng et al., J. Clin. Microbiol. 39(8): 2937-45, 2001; Komurian et al., J. Virol Methods 95(1-2): 111-9, 2001; Kleiber et al., J. Mol. Diagn 2(3): 158-66, 2000; Beames et al., J. Virol. 74(24): 11764-72, 2000; Kawai et al., J. Med. Virol. 58(2): 121-6, 1999; Mercier et al., J. Virol Methods 77(1): 1-9, 1999; Martell et al., J. Clin. Microbiol. 37(2): 327-32, 1999; Petrik et al., J. Virol Methods 64(2): 147-59, 1997; and Morris et al., J. Clin. Microbiol. 34(12): 2933-6, 1996.